WO2008029981A1 - Formulation de diagnostic pour la maladie de tsutsugamushi - Google Patents

Formulation de diagnostic pour la maladie de tsutsugamushi Download PDF

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Publication number
WO2008029981A1
WO2008029981A1 PCT/KR2007/001579 KR2007001579W WO2008029981A1 WO 2008029981 A1 WO2008029981 A1 WO 2008029981A1 KR 2007001579 W KR2007001579 W KR 2007001579W WO 2008029981 A1 WO2008029981 A1 WO 2008029981A1
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WIPO (PCT)
Prior art keywords
tsutsugamushi
protein
diagnostic kit
antigen
test
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PCT/KR2007/001579
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English (en)
Inventor
Yoon-Won Kim
Ik-Sang Kim
In-Ae Chang
Soo-Dong Woo
Young-Jin Kim
Ju-Me Chun
Woo-Chang Kim
Yong-Hwan Byun
Min-Kee Cho
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Immunemed, Inc.
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Application filed by Immunemed, Inc. filed Critical Immunemed, Inc.
Priority to CN2007800047896A priority Critical patent/CN101379398B/zh
Publication of WO2008029981A1 publication Critical patent/WO2008029981A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/29—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Richettsiales (o)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to a diagnostic kit for tsutsugamushi disease, in which antibodies against Orientia tsutsugamushi in a biological sample are detected using a mixed antigen of Orientia tsutsugamushi.
  • Tsutsugamushi disease is an acute febrile illness caused by Orientia tsutsugamushi, which belongs to scrub typhus group of the family Rickettsiaceae, and transmitted by the bite of larval- stage trombiculid mites. Symptoms, such as chills, fever, and headache occur in about 10 days (1 to 3 weeks) after being bitten by a larval-stage trombiculid mite, and a rash begins to appear on the trunk of a human body around one week after the onset of symptoms, and then spreads centrifugally to the extremities.
  • a Blister occurs at the bite site of larval-stage trombiculid mites, followed by ulcer formation with a size of 0.5 to 0.8 cm. The ulcer is covered by a black crust, which is referred to as eschar.
  • Some patients develop respiratory symptoms, in which chest x- rays showed pulmonary infiltration is present in approximately half of the patients. In some severe cases, the central nervous system may be infected, which leads to loss of consciousness or death.
  • Tsutsugamushi disease occurs worldwide, but is particularly frequent in Asia including Japan, China, Malaysia, Thailand, Vietnam, or the like (Tsutsugamushi disease in Korea, edited by WooHyun Jang, published by Seoheung Co., p. 21-27, 1994). The disease occurs most frequently in October, usually during the fall, September to early December (Kim, et al., Infection 20; 105-116, 1988). People working in the field or camping, may be at risk for acquiring tsutsugamushi disease.
  • Pathogenicity of Orientia tsutsugamushi is caused by systemic vasculitis due to attacking vascular endothelial cells. After invading to the blood, Orientia tsutsugamushi replicates in the vascular endothelial cells, which causes blood vessel damage, or microvascular thrombosis and inflammation around the blood vessels. Therefore, the disease is histopathologically characterized by that blood vessels of multiple organs are damaged, which may cause dermal necrosis, rash, meningitis, hearing loss, myocarditis, and heart failure. Further, a case of hypofibinogenemia due to intravascular coagulation has been reported.
  • Tsutsugamushi bacteria belonging to the same serotype are serologically associated with each other, and there is none or weak serologic cross- reactivity between the bacteria belonging to different serotypes.
  • Orientia tsut- sugamushi is classified into one species.
  • serotypes with different antigenicity there are many serotypes with different antigenicity, and they are classified into serotypes such as Gilliam, Karp, and Kato strains according to antigenicity.
  • Kangwon and Boryong strain which have different serologic reactivity from the known epidemic prototype strains, Gilliam and Karp, have been isolated.
  • Such serotype diversity depends on membrane protein antigens of Orientia tsut- sugamushi such as species-specific antigen and serotype-specific antigen.
  • Antigens were purified from Orientia tsutsugamushi, and then analyzed using polyacrylamide gel electrophoresis and immunoblotting. Consequently, it was found that major antigens with the size of 70 kDa, 60 kDa, 54 to 56 kDa, and 46 to 47 kDa are present (Takahashi K, et al., Microbiol. Immunol.
  • each antigen with the size of 46 to 47 kDa, 54 to 56 kDa, and 70 kDa has strong antigenicity
  • each antigen with the size of 70 kDa, and 46 to 47kDa is a strain-specific antigen (Tamura A, et al., Microbiol. Immunol. 26: 321, 1982).
  • a diagnostic kit for tsutsugamushi disease including a test strip comprising a mixed antigen of tsut- sugamushi.
  • Fig. 1 is a photograph showing the results of 10% SDS-PAGE to confirm a process for purifying an expressed, recombinant antigen protein.
  • Fig. 2 is a photograph showing the reactivity between a protein (mixture of cr56, r21 and kr56) purified from transformed E. coli and sera from patients using a dot-blot immunoassay in order to confirm its suitability as an antigen for diagnosis.
  • FIG. 3 illustrates the structure of a test strip in the diagnostic kit according to the present invention manufactured by introducing an immunochromatography technique.
  • FIG. 4 illustrates the structure of a final sample of diagnostic kit capped with a plastic and a method for analyzing its results in the diagnostic kit of the present invention.
  • the present invention relates to a diagnostic kit for tsutsugamushi disease including a test strip comprising a mixed antigen of tsutsugamushi.
  • the present invention relates to a diagnostic kit for tsutsugamushi disease including a test strip to detect antibodies against Orientia tsutsugamushi in a biological sample, in which a test strip comprises:
  • test membrane comprising a test line containing a mixed antigen of tsutsugamushi and a control line containing a control protein; and [21] (d) an absorption pad absorbing the residual sample.
  • a diagnostic kit of the invention for tsutsugamushi disease preferably includes a test strip comprising a sample pad, a gold conjugation pad, a test membrane, and an absorption pad.
  • the sample pad is superposed on the gold conjugation pad to form a first overlap portion
  • the gold conjugation pad is superposed on the membrane to form a second overlap portion.
  • the membrane and the absorption pad form a third overlap portion.
  • the gold-labeled gold particle has preferably a diameter of 20 to 55 nm, more preferably 20 to 40 nm, and works as an indicator dye. While the biological sample migrates through the gold conjugation pad, antibodies in the sample bind with the gold-labeled protein A or the like to form a complex, and the complex migrates along the membrane.
  • biological sample means a serum or blood plasma from mammals including human suspected of having tsutsugamushi disease.
  • the biological sample can be diluted or undiluted prior to adding dropwise to the sample pad of the diagnostic kit of the invention.
  • tsutsugamushi antibodies are present in the biological sample, while the sample migrates through the test line comprising a mixed antigen of tsutsugamushi in the diagnostic kit, the antibodies binds with the mixed antigens of tsutsugamushi to make color change on the test line, which can be observed with the naked eye.
  • the mixed antigen of tsutsugamushi cannot bind with the antibodies not to make color change on the test line.
  • the tsutsugamushi antibodies bind to the mixed antigen of tsutsugamushi immobilized in the test line on the membrane.
  • This antigen- antibody complex is formed as an indicator dye by the gold particle.
  • the red purple band is observed, which indicates a positive response, that is, the presence of tsutsugamushi antibody in the biological sample.
  • mixed antigen of tsutsugamushi means a mixture of a recombinant antigen protein derived from Orientia tsutsugamushi Gilliam, Karp, and Kato strains, an antigen protein derived from Kangwon strains, and an antigen protein derived from Boryong strain, in which each strain causes tsutsugamushi disease.
  • the mixed antigen of tsutsugamushi is a mixture of a chimeric recombinant protein derived from recombinant fusion gene, in which genes coding for epitopes of antigen of Orientia tsutsugamushi Gilliam, Karp, and Kato strains are connected in series, and each recombinant protein derived from gene coding for antigen of Orientia tsutsugamushi Kangwon and Boryong strains.
  • a fusion protein antigen can be produced, isolated, and purified by a one-step process, in which genes coding for 56 kDa protein fragments of standard serotype Gilliam, Karp, and Kato strains are amplified by polymerase chain reaction (PCR), the amplified DNA fragments are connected in series, the recombinant DNA is cloned into a protein expressing vector to express in E. coli, and the expressed protein is isolated and purified to be used as an antigen for diagnosing tsutsugamushi disease, on the basis of Korean Patent Application No. 2002-0020281, which discloses that 56 kDa protein of Orientia tsutsugamushi has antigenicity, thereby being usefully used as a diagnostic protein.
  • PCR polymerase chain reaction
  • Kato strains a gene coding for a 56 kDa antigenic protein Of Orientia tsutsugamushi Kangwon strain and a gene coding for 21 kDa antigenic protein Of Orientia tsutsugamushi Boryong strain are amplified by PCR, the amplified DNA fragments are cloned into a protein expressing vector to express in E.
  • the expressed proteins are isolated and purified to use as a diagnostic proteins with the 56 kDa chimeric recombinant protein derived from Orientia tsutsugamushi Gilliam, Karp, and Kato strains, in order to improve diagnostic sensitivity for tsutsugamushi disease.
  • a 56 kDa recombinant chimeric protein of Orientia tsutsugamushi Gilliam, Karp, and Kato strains a 56 kDa recombinant protein of Orientia tsutsugamushi Kangwon strain, and a 21 kDa recombinant protein of Orientia tsutsugamushi Boryong strain are prepared at a concentration of 1 mg/ml, respectively, and they are then mixed at a volume ratio of 5:2: 1. 1 mg/ml of the mixed antigen of tsutsugamushi is added dropwise on the test line.
  • the positive result for tsutsugamushi disease is a case that a color is developed on the test line, in addition to the control line.
  • the control line is to confirm whether the diagnostic kit works or not.
  • a color is developed only on the control line.
  • the control line contains a control protein binding with a gold-labeled antibody. Examples thereof include a rabbit anti-goat IgG polyclonal antibody, a goat anti-human IgG polyclonal antibody or a goat anti-human IgM polyclonal antibody.
  • the control line may contain 0.1 mg/ml to 1 mg/ml of the control proteins.
  • the diagnostic kit of the invention further comprises a pre-test line containing an extract of E. coli not expressing the antigen protein of tsutsugamushi.
  • the pre-test line in the diagnostic kit of the invention using the antigen protein of Orientia tsutsugamushi derived from E. coli, allows to remove any possibility of misdiagnosis by non specific result from the reaction of antibodies which are derived from E. coli in human, on the protein of E. coli contaminated in the purified tsutsugamushi antigen protein.
  • the pre-test line may contain the extract of E. coli at a concentration of 0.5 mg/ml to 0.6 mg/ml.
  • the test membrane can contain the suitable materials such as nitrocellulose membrane (MiliporeTM XA3J072100) with a standard size of 10 x 500 nm.
  • the pre-test line, the test line, and the control line are preferably arranged in this order.
  • the pre-test line, the test line, and the control line are arranged with an interval of 2.0 to 4.5 mm, preferably 2.5 to 3.0 mm.
  • the gold conjugation pad can contain the suitable detecting markers.
  • the marker include a gold-labeled protein A, a gold-labeled anti-human IgG antibody, and a gold-labeled anti-human IgM antibody.
  • the gold conjugation pad is preferably placed adjacent to the sample pad. In the case where the gold conjugation pad contains an excessive amount of the gold-labeled marker, the excessive amplified signals, which are generated in a lower limit concentration of the tsutsugamushi antibody, increase to cause a false-positive result.
  • the gold-colloid solution is diluted to be optical density (OD) of 1 to 6, preferably 2 to 4.
  • the absorption pad is placed at the opposite end point of the membrane and absorbs the biological sample, such as serum or blood plasma, migrating along the membrane by capillary action.
  • the diagnostic kit of the invention can comprise a test strip.
  • the diagnostic kit of the invention can comprise two strips of a first strip and a second strip branched from a sample pad.
  • the first strip can be used for detecting human IgG antibody
  • the second strip can be used for detecting a human IgM antibody against the antigen protein of tsutsugamushi.
  • the first strip may comprise the gold conjugation pad containing a goat anti-human IgG antibody or protein A and the control line containing a rabbit anti-goat IgG polyclonal antibody.
  • the second strip may comprise the gold conjugation pad containing a goat anti-human IgM antibody and the control line containing a goat anti- human IgM polyclonal antibody.
  • the first strip and the second strip can be preferably arranged in parallel, but are not limited thereto.
  • the first strip and the second strip can be connected in series to arrange in opposite direction or in various directions.
  • test strip can be packed with a surrounding case in order to assure the safety of each diagnostic kit of the invention.
  • the diagnostic kit can be stored at room temperature for a long period of time (at least 12 months or longer) without any loss of diagnostic activity.
  • diagnosis takes about 5 to 10, not exceeding 15 minutes.
  • the diagnostic result indicates whether a patient is infected with tsutsugamushi. For example, if the biological sample is collected from the patient infected with tsutsugamushi, the diagnostic kit of the invention gives a positive response, whereby the patient can be diagnosed as having tsutsugamushi disease. If the biological sample is collected from the patient not infected with tsutsugamushi, the diagnostic kit of the invention gives a negative response, whereby the patient can be diagnosed as not having tsutsugamushi disease.
  • the serum displaying very weak positive signal was tested using the kit comprising the above proteins and the 56 kDa protein derived from Orientia tsutsugamushi Kangwon strain, the positive signal became strong, that is, its sensitivity was improved (100%). Accordingly, the mixed antigen of tsutsugamushi used in the invention was found to have excellent sensitivity and specificity.
  • the present invention provides a diagnostic composition for tsutsugamushi disease comprising a 56 kDa fusion protein derived from Orientia tsutsugamushi Gilliam, Karp, and Kato strains, the 56 kDa protein derived from Orientia tsutsugamushi Kangwon strain, and the 21 kDa protein derived from Orientia tsutsugamushi Boryong strain as a mixed antigen of tsutsugamushi.
  • the 56 kDa protein derived from Orientia tsutsugamushi Kangwon strain in the mixed antigen of tsutsugamushi has an amino acid sequence of SEQ ID NO: 6
  • the 21 kDa protein derived from Orientia tsutsugamushi Boryong strain has an amino acid sequence of SEQ ID NO: 3.
  • the method for detecting antibody against tsutsugamushi disease using the above described diagnostic kit for tsutsugamushi disease is as follows. First, when a biological sample to be analyzed is added dropwise to the sample pad of the diagnostic kit, which is a portion absorbing the sample, the biological sample migrates to the gold conjugation pad by capillary action.
  • the antibody in the biological sample binds to the marker in the gold conjugation pad, for example, gold-labeled protein A, gold-labeled anti-human IgG antibody, or gold-labeled anti-human IgM antibody, so as to form a colloid.
  • the biological sample for analysis is not immobilized in the gold conjugation pad, but continues to migrate to the test line, in which the mixed antigen of tsutsugamushi is immobilized. In the case of the sample from a patient infected with tsutsugamushi disease, the sample reacts with the mixed antigen of tsutsugamushi to produce antigen-antibody reaction.
  • the diagnostic kit of the invention is performed using immunochromatography by antigen- antibody reaction, whereby the result can be observed with the naked eye without any equipment.
  • the pre-test line of the diagnostic kit eliminates any possibility of misdiagnosis, which may be presented due to the presence of antibodies against E. coli in human serum instead of Orientia tsutsugamushi, thereby improving the accuracy of the diagnosis for tsutsugamushi disease.
  • the chimeric recombinant protein (cr56) comprising the major epitopes of Orientia tsutsugamushi Gilliam, Karp, and Kato strains was expressed, isolated, and purified in the same manners as Examples disclosed in Korean Patent No. 2002-0020281.
  • Orientia tsutsugamushi Gilliam, Karp, and Kato strains were cultured in mouse L-929 cells, collected and purified. The purified cells were digested with enzymes, and then their DNA was extracted with phenol and ethanol.
  • each DNA portion having 30% or more amino acid sequence homology among Boryong and Gilliam, Karp, and Kato serotypes was selected, and a pair of oligonucleotide primer for each Gilliam, Karp, and Kato strain was prepared.
  • PCR was carried out with each primer and the purified DNA of Orientia tsutsugamushi as a template to amplify DNA fragments of Gilliam, Karp, and Kato strains.
  • the PCR products were purified and cloned to be ligated at restriction enzyme digestion sites of a pTYB12 vector.
  • the DNA fragment of Gilliam strain was introduced into a pTYB12 vector to prepare a vector, pTG3, and then the DNA fragment of Karp strain was introduced into the pTG3 vector to prepare a vector, pTGPl.
  • the DNA fragment of Kato strain was introduced into the pTGPl vector to prepare a vector, pTGPT2.
  • the DNA fragment connected with the DNA sequences of Gilliam, Karp, and Kato strains in series was cut from the pTGPT2 vector.
  • the DNA fragment was introduced into a pET22b(+) vector to prepare a vector, pETb7.
  • E. coli was transformed by the prepared expression vector, and then the transformed E. coli was cultured to induce expression of the fusion protein.
  • the fusion protein was confirmed using electrophoresis and western blotting, isolated and purified.
  • a gene having species-specific antigenicity in Orientia tsutsugamushi Boryong strains was expressed in E. coli to isolate and purify the recombinant protein (r21) as the following method.
  • Plasmid pET22b expresses a His-Tagged fusion protein [60] [61] (3) Gene cloning [62] The DNA amplified by polymerase chain reaction was separated by electrophoresis with 1.2% agarose gel, and then recovered using a QIAGEN gel elution kit (QIAGEN Inc., USA). The amplified DNA band with a right size was cut on a UV transil- luminator. The band was transferred to a microtube and completely dissolved with a NaI solution (three volume of the agarose gel) at 6O 0 C for 5 minutes. Glass milk was added thereto, and the microtube was vortexed for 10 seconds.
  • the solution was centrifuged at 17,000 g (Hanil, AI.5S-24, 12,000rpm) at room temperature for 1 minute to remove a supernatant.
  • An elution buffer was added to the residual solution, and then the microtube was vortexed to centrifuge at 17,000 g (12,000 rpm). A new supernatant was transferred to a new microtube. 40ng of the purified DNA was cloned into the pET22b(+) vector.
  • E.coli BL21 cells cultured in LB medium for 18 hours were diluted to 100-fold with LB medium.
  • E.coli BL21 cells were recultured to be an absorbance of 0.3 at 600 nm. After placed in ice for 30 minutes, the supernatant was discarded.
  • 0.1M CaCl was added to be half volume of the medium, and then the bacteria were suspended. The suspended bacteria were placed in ice for 1 hour, and centrifuged at 2,000 g (4,100 rpm) for 10 minutes. The pellet was suspended in 0.1M CaCl with one tenths volume of the culture medium to be used for the transformation.
  • the ligation product prepared in the above and the competent cells were mixed to be placed in ice, and then heat shocked at 42 0 C.
  • the LB medium was added thereto, and cultured at 37°C for 1 hour.
  • the culture medium was spread onto an LB agar plate to culture at 37°C.
  • the transformed E.coli BL21 cells were cultured at 37°C for 18 hours, and then diluted about 100-fold to be OD600 of 0.5. IPTG was added thereto to be a concentration of 0.3 mM, and then cultured for 2 hours.
  • the transformed E.coli BL21 cells were centrifuged at 5,800 g (7,000 rpm). The pellet was suspended in a 1 X binding buffer (5 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl) to sonicate with an ul- trasonicator at 18 Hz for 15 seconds six times. The sonicated solution was centrifuged at 23,000 g (14,000 rpm). The supernatant was used as a soluble fraction, and the pellet was used as an insoluble fraction. SDS-PAGE was carried out with each fraction to confirm which fraction has the recombinant protein.
  • a column was filled with 2 ml of Hisbind resin (Novagen), and then prepared with distilled water, a 1 X charge buffer, and a 1 X binding buffer. After the antigen protein was passed through the column, the column was washed with a 1 X washing buffer. An elution buffer (100 mM to 300 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl pH 7.9) was passed through the column to recover each 1 ml of the protein solution. A concentration of the protein in each fraction was determined by the Lowry assay. SDS- PAGE and western blotting were carried out to confirm the protein (SEQ ID NO: 3) (Fig. 1).
  • Plasmid pET30a expresses a His-Tagged fusion protein [84] [85] (2) Gene cloning and expression [86] The DNA amplified by polymerase chain reaction (362 amino acids of amino acid 84 (250 bp) to 445 (1,335 bp) (total 1,086 bp)) was separated by electrophoresis with 1.2% agarose gel, and then recovered using a QIAGEN gel elution kit (QIAGEN Inc., USA). The amplified DNA band with a right size was cut on a UV transilluminator.
  • the band was transferred to a microtube, and completely dissolved with a NaI solution (three volume of the agarose gel) at 6O 0 C for 5 minutes. Glass milk was added thereto, and the microtube was vortexed for 10 seconds. The solution was centrifuged at 17,000 g (Hanil, AI.5S-24, 12,000rpm) at room temperature for 1 minute to remove a supernatant. The microtube was left at room temperature for 5 minutes, and dried. An elution buffer was added thereto, and then the microtube was vortexed to centrifuge at 17,000 g (12,000 rpm). A new supernatant was transferred to a new microtube.
  • a NaI solution three volume of the agarose gel
  • the purified PCR product and pET30a vector were digested with the restriction enzymes, Nco I and Sal I, and then recovered by the same method using Geneclean kit II. 100 ng of the restricted vector and 100 ng of the restricted PCR product were mixed with each other, and then the mixture of 10 X ligase buffer (250 mM Tris-HCl pH 7.8, 100 mM MgCl , 20 mM DTT and 4 mM ATP) and ligase was added thereto, and then subjected to reaction at 4°C for 18 hours.
  • 10 X ligase buffer 250 mM Tris-HCl pH 7.8, 100 mM MgCl , 20 mM DTT and 4 mM ATP
  • E.coli BL21 cells cultured in LB medium for 18 hours were diluted to 100-fold with
  • E.coli BL21 cells were recultured to be an absorbance of 0.3 at 600 nm. After placed in ice for 30 minutes, the supernatant was discarded.
  • 0.1M CaCl was added to be half volume of the medium, and then the bacteria were suspended. The suspended bacteria were placed in ice for 1 hour, and centrifuged at 2,000 g (4,100 rpm) for 10 minutes. The pellet was suspended in 0.1M CaCl with one tenths volume of the culture medium to be used for the transformation.
  • the supernatant was transferred into a binding column tube, and then centrifuged at 17,000 xg (12,000 rpm) at room temperature to remove the extracted solution. 80% ethanol was added thereto, centrifuged, and then the binding column was only placed in a new tube. An elution buffer was added thereto, left at room temperature, and then centrifuged to transfer a supernatant into a new microtube.
  • the transformed E.coli BL21 cells were cultured at 37°C for 18 hours, and then diluted about 100-fold to be OD600 of 0.5. IPTG was added thereto to be a concentration of 0.3 mM, and then cultured for 2 hours.
  • the transformed E.coli BL21 cells were centrifuged at 5,800 g (7,000 rpm). The pellet was suspended in a 1 X binding buffer (5 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl) to sonicate with an ul- trasonicator at 18 Hz for 15 seconds six times. The sonicated solution was centrifuged at 23,000 g (14,000 rpm). The supernatant was used as a soluble fraction, and the pellet was used as an insoluble fraction. SDS-PAGE was carried out with each fraction to confirm which fraction has the recombinant protein.
  • a column was filled with 2 ml of Hisbind resin (Novagen), and then prepared with distilled water, a 1 X charge buffer, and a 1 X binding buffer. After the antigen protein was passed through the column, the column was washed with a 1 X washing buffer. An elution buffer (300 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl pH 7.9) was passed through the column to recover each 1 ml of the protein solution. A concentration of the protein in each fraction was determined by the Lowry assay. SDS-PAGE and western blotting were carried out to confirm the protein (SEQ ID NO: 6) (Fig. 1).
  • Example 2 Diagnostic analysis of purified antigen protein using ELISA.
  • the present test was performed in order to assure the possibility of diagnosing tsut- sugamushi disease by antigen-antibody reaction with the antibody in the serum from the patient with tsutsugamushi disease, using the cr56 protein, which was expressed in E.coli by fusion of three prototypes of Orientia tsutsugamushi, and the kr56 protein and the r21 protein, which were respectively expressed by two serotypes, Kangwon and Boryong.
  • the purified protein antigens were diluted with a 0.05 M carbonate buffer (pH 9.6) to be as Table 3.
  • a 96 well plate was coated with the diluted antigen, and then subjected to reaction for 18 hours.
  • the plate was washed with a phosphate buffer (PBS) containing 0.05% Tween 20 twice, and then 100 ⁇ l of 3% bovine serum albumin was added to the wells to block at 37 0 C for 2 hours.
  • PBS phosphate buffer
  • a 2-fold serial dilution of a patient's serum was preformed. 100 ⁇ l of the diluted serum was used as a primary antibody.
  • a peroxidase conjugated goat anti-human IgG was diluted to 1:6,000 to use as a secondary antibody.
  • Each antigen protein (cr56 and r21) was diluted to be 1.25 ⁇ g/ml.
  • the present test was performed in order to assure the possibility of diagnosing tsut- sugamushi disease by antigen-antibody reaction with the antibody in the serum from the patient with tsutsugamushi disease, using the cr56 protein, which was expressed in E.coli by fusion of three prototypes of Orientia tsutsugamushi, and the kr56 protein and the r21 protein, which were respectively expressed by two serotypes, Kangwon and Boryong. 1 ⁇ g of the purified protein antigen (1 ⁇ g/ ⁇ l) was added dropwise to a nitrocellulose membrane, and then dried at 37 0 C for 1 hour.
  • the dried membrane was blocked with a TBST (Tris-buffered saline containing 0.5% Tween 20) solution containing 5% skim milk for 1 hour.
  • the patient's serum was diluted to 1:3,000 as a primary antibody, and then added to the membrane. The reaction was performed for 1 hour.
  • a peroxidase conjugated goat anti-human IgG was used as a secondary antibody. The result is shown in Fig. 2 and Table 4.
  • the mixed antigen of cr56, r21, and kr56 proteins was found to have the highest sensitivity and specificity.
  • the antigen was purified using a His-bind affinity chromatography.
  • the protein derived from E.coli could not be completely removed. That is, it was difficult to remove completely the protein derived from E.coli by purification. Therefore, E.coli host cell, which does not express the antigen protein of tsutsugamushi, was cultured to extract. The protein of extracted host cell was sprayed for the pre-test line before the test line, thereby human antibody reacting with the protein derived from E.coli. was removed.
  • Example 5 Production of stick type sample of diagnostic kit
  • a test strip sample for diagnosis was manufactured using a dispenser purchased from NANO ENG Co.
  • the test strip as shown in Fig. 3, consisted of a nitrocellulose membrane, a sample pad, an absorption pad, and a gold conjugation pad (glass fiber).
  • the nitrocellulose membrane was purchased from Millipore to use. In order to avoid the membrane damage, a part of the membrane was attached with a transparent plastic. Three proteins were sprayed on the nitrocellulose membrane.
  • One of three proteins is the mixed protein, in which cr56, kr56, and r21 antigen proteins expressed in E.coli were mixed with the suitable amount, to detect the antibody against tsutsugamushi in patient's serum, and used as a test line. Another one of three proteins is used as a control line to confirm whether the developing system works well or not.
  • the other one of three proteins is the E.coli extract not expressing the antigen protein of tsutsugamushi, and used as a pre-test line to remove human antibody reacts with the protein derived from E.coli. Each protein was sprayed on the pre-test line, the test line, and the control line with the suitable amount using the dispenser, and then dried at 37 0 C for 2 hours.
  • the glass fiber was absorbed with the colloidal gold conjugate in a 5% trehalose solution, and then dried at a vacuum dry oven for 2 hours.
  • the sample pad and the absorption pad were cut with the predetermined size.
  • the sample pad was immersed in a 250 mM Tris solution containing 1% tween 20 in order to absorb the serum or blood plasma well, and then completely dried at 37 0 C for 4 hours.
  • the gold conjugation pad (glass fiber) sprayed with the colloidal gold conjugates was placed below the nitrocellulose membrane, and the sample pad was placed therebelow.
  • the absorption pad was placed above the nitrocellulose membrane.
  • the final diagnostic stick was cut with a width of 4 mm and a height of 60 mm.
  • the proteins used herein are shown in Table 5.
  • the samples 1 and 2 are able to detect only IgG against the antigen protein of tsutsugamushi in the human serum, and the samples 3 and 4 are able to detect only IgM. Therefore, with respect to its use, the samples 1 and 2 can be diagnostic tool to detect middle-late stage patients and the samples 3 and 4 can be a useful diagnostic tool to detect early stage patients.
  • two strips were placed in one plastic, so as to diagnose simultaneously. The efficacy test was performed for the sample of the diagnostic kit using sera from patient and healthy person.
  • the sample of the diagnostic kit was found to be fast, accurate, and simple diagnostic kit for tsutsugamushi disease. Further, the cross-reactivity was not found to occur from the test result using sera from patients with other acute febrile illness (haemorrhagic fever with renal syndrome, murine typhus, leptospirosis). Accordingly, the diagnostic kit in the invention was found to have more improved sensitivity than the conventional kit. [132] [133] [Table 5] [134] Protein used for manufacturing sample of diagnostic kit [135]
  • test strips manufactured herein were placed in one plastic device to produce the final diagnostic kit as shown in Fig. 4.
  • the manual is as follows.
  • the plasma blood or serum from the patient was diluted with a suitable solution. 400 ⁇ l of the diluted plasma blood or serum was added drop wise to a small well on the bottom to left for 10 minutes. It is observed whether red purple color is developed in the test line (position marked as T) and the control line (position marked as C) or not. In the case where red purple color is developed in both of two lines, the result is positive. In the case where red purple color is only developed in the control line, the result is negative.
  • Example 6 Efficacy test for strip sample of diagnostic kit [144] In order to confirm the efficacy of the manufactured strip sample of the diagnostic kit, the test was performed using sera from 65 persons containing patients with tsut- sugamushi, healthy persons, and patients with other acute febrile illness. The manual is as follows. The sera from the patients or healthy persons were diluted with a suitable solution. 400 ⁇ l of the diluted sera were added dropwise to a small well on the bottom and it is left for 10 minutes. It is observed whether red purple color is developed in the test line (position marked as T) and the control line (position marked as C) or not. In the case where red purple color is developed in both of two lines, the result is positive.
  • the diagnosis is performed using proteins, which have antigenicity of Gilliam, Karp, and Kato, Kangwon and Boryong strains of Orientia tsutsugamushi by immunochro- matography method. Therefore, the diagnostic result can be observed simply, accurately and fast with the naked eye, as compared to the conventional im- munofluorescent antibody technique or the like.

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Abstract

Kit de diagnostic pour la maladie de tsutsugamushi faisant appel à un antigène mélangé de tsutsugamushi. Plus précisément, ce kit comprend un bâtonnet diagnostique qui comporte une tampon à échantillon absorbant l'échantillon, un tampon de conjugués or se liant avec les anticorps dans l'échantillon, une membrane d'essai qui comporte une ligne d'essai contenant un antigène mélangé de tsutsugamushi et une ligne témoin contenant une protéine témoin, un tampon d'absorption absorbant l'échantillon résiduel. Dans ce kit, l'anticorps vis-à-vis du tsutsugamushi peut être simplement détecté, sans utilisation d'équipement, et l'infection par Orientia tsutsugamushi peut ainsi être confirmée à l'oeil nu.
PCT/KR2007/001579 2006-09-04 2007-03-30 Formulation de diagnostic pour la maladie de tsutsugamushi WO2008029981A1 (fr)

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KR20170127833A (ko) 2016-05-13 2017-11-22 광주과학기술원 진단용 키트
WO2018185487A1 (fr) * 2017-04-07 2018-10-11 Robert Graham Price Test spécifique, rapide de différenciation de bactéries à gram positif et à gram négatif

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